Sepsis is a serious medical condition caused by an overwhelming response of the host immune system to infection. It can trigger widespread inflammation, which can give rise to impaired blood flow. As sepsis progresses, the body's organs can be starved for oxygen and nutrients, causing permanent damage and eventual failure. Left improperly diagnosed or otherwise untreated, the heart may weaken and septic shock can occur, leading to multiple organ failure and death. Blood cultures are required to detect the presence of bacteria or yeast in the blood of sepsis patients. If a microorganism is present, (positive blood culture (“PBC”)) the microorganism(s) must be identified and antibiotic susceptibility determined in order to provide appropriate treatment. The PBC samples are used to isolate, identify and perform antimicrobial susceptibility testing (“AST”). The microorganism(s) are often identified by methods such as mass spectrometry, including MALDI-TOF/MS or phenotypic growth-based methods, such as Phoenix™ ID.
In order to identify the microorganism(s), perform phenotypic analysis on the microorganism, and perform AST testing, intact, viable microorganism(s) need(s) to be isolated from the blood cells and other material in the collected sample. For identification of the microorganism by mass spectrometry, the microbial sample needs to be sufficiently free from substances known to interfere with MALDI-TOF/MS identification, such as blood cell components, other cellular debris, and salts. In addition, the microbial sample needs to be of sufficient quantity in order to obtain a reliable identification. Phenotypic identification methods, such as Phoenix™ ID, require intact, viable microorganism free from substances that may interfere with the enzymatic substrates of the assay. For AST testing, such as Phoenix™ AST, the microbial sample needs to contain viable, unaltered microorganism capable of growth in the presence of antibiotic, if resistance mechanisms are present, during performance of the assay. It is important for all methods to be of sufficient quantity and purity as carryover of residual blood or media components will interfere either directly or by falsely increasing the concentration (turbidity) of microorganism.
Current techniques for isolating viable microorganism from a PBC sample include sub-culturing the microorganism(s), which can take up to 72 hours. This results in the delay of treatment or treatment with inappropriate antibiotics.
Certain strains of microorganisms are particularly difficult to isolate from a PBC sample while maintaining viability of the organism, such as, for example, Streptococcus pneumoniae (S. pneumoniae). Part of this difficulty is traced to the activation of autolysin by S. pneumoniae which causes the microbial cells to “self-destruct”. See “Streptococcus pneumoniae Antigen Test Using Positive Blood Culture Bottles as an Alternative Method To Diagnose Pneumococcal Bacteremia”, Journal of Clinical Microbiology, Vol. 43, No. 5, May 2005, p. 2510-2512. The current method for isolating microorganisms from septic patients, including, S. pneumoniae, includes inoculating blood culture bottles. Once a positive signal is achieved, a portion of the PBC sample is removed to perform a gram stain and another portion is used to sub-culture the microorganism. Microbial colonies from the sub-culture are used to perform downstream testing such as identification by MALDI-TOF/MS, phenotypic identification methods, and AST testing.
Additional techniques for isolating viable microorganism(s) from a PBC sample often utilize liquid separation methods containing lysis buffers with detergents that lyse the blood cells in the PBC sample. After lysis, the lysed blood cells can be removed while the microorganism(s) is/are retained. However, the use of these lysis buffers often result in compromised, damaged, or non-viable microorganism(s) which is/are insufficient for performing certain growth-based identification methods such as AST testing.
One such liquid separation method, the Bruker Sepsityper™ system, allows for direct testing of the microorganism from a PBC sample by MALDI-TOF/MS without the need for sub-culturing the microorganism. This method uses Sodium Dodecyl Sulfate (“SDS”) and centrifugation to generate a pellet of microbes. While the Sepsityper™ method will generally support MALDI-TOF/MS testing of the microbial pellet, there is insufficient viability to support growth-based identification methods and AST methods, due to the interaction of the harsh detergents on the microbial cell wall.
Prod'hom et al., “Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Direct Bacterial Identification from Positive Blood Culture Pellets”, Journal of Clinical Microbiology, Vol. 48, No. 4, p. 1481-1483 (Feb. 17, 2010). discloses a method for lysing erythrocytes in a PBC sample using ammonium chloride to prepare a bacterial pellet for MALDI-TOF/MS analysis. However, these methods are insufficient in obtaining reliable MALDI-TOF/MS data across a panel of microorganism, including S. pneumoniae. In addition, there is no indication that these methods will maintain sufficient viability of the microorganism for use in growth-based identification methods and AST testing.
Hansson et al., “Microfluidic Blood Sample Preparation for Rapid Sepsis Diagnostics”, KTH Engineering Sciences, (2012) suggests the use of detergents for lysing blood cells and selectively lysing certain types of blood cells with ammonium chloride. However, this reference is silent with regard to specific formulations or methods that would allow for the isolation of viable microorganism from a PBC sample that is free from interfering substances and would allow for multiple downstream testing from one PBC sample, such as both MALDI-TOF/MS identification and AST testing.
Both M. Drancourt, “Detection of Microorganisms in Blood Specimens Using Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry: A Review, Clinical Microbiology and Infection, 16:1620-1625, (2010) and WO 2010/100612 to Nassif et al. describe methods for isolating microorganism from a PBC sample which include removing red blood cells from a PBC sample by adding saponin and/or ammonium chloride to a portion of the PBC sample, centrifuging the mixture, and washing the resulting pellet with water to remove residual blood proteins. However, these methods produce inconsistent identification of the microorganism at the species level across a panel of microorganisms and/or fail to identify S. pneumoniae. Furthermore, there is no indication that these methods result in a microbial pellet with sufficient viability to perform growth-based testing methods, such as AST.
Accordingly, it is desirable to develop reagents and methods that rapidly separate microorganism(s) from a PBC sample while maintaining the viability of the microorganism(s), so that analytical growth-based methods that require cell viability, such as AST testing, can be performed. Additionally, it is desirable that the reagents and methods can be used to isolate viable cells from all types of microorganism, including S. pneumoniae. 